snRNAs from Radical Prostatectomy Specimens Have the Potential to Serve as Prognostic Factors for Clinical Recurrence after Biochemical Recurrence in Patients with High-Risk Prostate Cancer

Simple Summary In patients with high-risk prostate cancer (HRPC) after radical prostatectomy, biochemical recurrence increases the risk of distant metastasis. Therefore, complementary prognostic biomarkers are required to identify the subpopulation of patients with HRPC who develop clinical recurrence after biochemical recurrence. This study was performed to identify prognostic factors for clinical recurrence in patients with HRPC who experience biochemical recurrence by conducting an analysis of the expression levels of snRNAs in formalin-fixed paraffin-embedded (FFPE) radical prostatectomy samples. The FFPE sample-derived snRNA RNU1-1/RNU1-2 could serve as an independent prognostic factor of clinical recurrence-free survival after biochemical recurrence of HRPC cases where known prognostic factors (e.g., Gleason score) cannot distinguish between patients with clinical and non-clinical recurrence. Thus, snRNAs associated with prostate cancer may assist the early detection of clinical recurrence in patients with HRPC, allowing for more tailored and restorative treatments. Abstract In patients with high-risk prostate cancer (HRPC) after radical prostatectomy (RP), biochemical recurrence (BCR) increases the risk of distant metastasis. Accordingly, additional prognostic biomarkers are required to identify the subpopulation of patients with HRPC who develop clinical recurrence (CR) after BCR. The objective of this study was to identify biomarkers in formalin-fixed paraffin-embedded (FFPE) RP samples that are prognostic for CR in patients with HRPC who experience BCR after RP (post-RP BCR). First, we performed a preliminary RNA sequencing analysis to comprehensively profile RNA expression in FFPE RP samples obtained from patients with HRPC who developed CR after post-RP BCR and found that many snRNAs were very abundant in preserved FFPE samples. Subsequently, we used quantitative polymerase chain reaction (qPCR) to compare the expression levels of highly abundant snRNAs in FFPE RP samples from patients with HRPC with and without CR after post-RP BCR (21 CR patients and 46 non-CR patients who had more than 5 years of follow-up after BCR). The qPCR analysis revealed that the expression levels of snRNA RNU1-1/1-2 and RNU4-1 were significantly higher in patients with CR than in patients without CR. These snRNAs were significantly correlated with clinical recurrence-free survival (RFS) in patients with HRPC who experienced post-RP BCR. Furthermore, snRNA RNU1-1/1-2 could serve as an independent prognostic factor for clinical RFS in post-RP BCR of HRPC cases where known prognostic factors (e.g., Gleason score) cannot distinguish between CR and non-CR patients. Our findings provide new insights into the involvement of snRNAs in prostate cancer progression.


Simple Summary:
In patients with high-risk prostate cancer (HRPC) after radical prostatectomy, biochemical recurrence increases the risk of distant metastasis.Therefore, complementary prognostic biomarkers are required to identify the subpopulation of patients with HRPC who develop clinical recurrence after biochemical recurrence.This study was performed to identify prognostic factors for clinical recurrence in patients with HRPC who experience biochemical recurrence by conducting an analysis of the expression levels of snRNAs in formalin-fixed paraffin-embedded (FFPE) radical prostatectomy samples.The FFPE sample-derived snRNA RNU1-1/RNU1-2 could serve as an independent prognostic factor of clinical recurrence-free survival after biochemical recurrence of HRPC cases where known prognostic factors (e.g., Gleason score) cannot distinguish between patients with clinical and non-clinical recurrence.Thus, snRNAs associated with prostate cancer may assist the early detection of clinical recurrence in patients with HRPC, allowing for more tailored and restorative treatments.

Abstract:
In patients with high-risk prostate cancer (HRPC) after radical prostatectomy (RP), biochemical recurrence (BCR) increases the risk of distant metastasis.Accordingly, additional prognostic biomarkers are required to identify the subpopulation of patients with HRPC who develop clinical recurrence (CR) after BCR.The objective of this study was to identify biomarkers in formalin-fixed paraffin-embedded (FFPE) RP samples that are prognostic for CR in patients with HRPC who experience BCR after RP (post-RP BCR).First, we performed a preliminary RNA sequencing analysis to comprehensively profile RNA expression in FFPE RP samples obtained from patients with HRPC who developed CR after post-RP BCR and found that many snRNAs were very abundant in preserved FFPE samples.Subsequently, we used quantitative polymerase chain reaction (qPCR) to compare the expression levels of highly abundant snRNAs in FFPE RP samples from patients with HRPC with and without CR after post-RP BCR (21 CR patients and 46 non-CR patients who had more than 5 years of follow-up after BCR).The qPCR analysis revealed that the expression levels of snRNA RNU1-1/1-2 and RNU4-1 were significantly higher in patients with CR than in patients without CR.These snRNAs were significantly correlated with clinical recurrence-free survival (RFS) in patients with HRPC who experienced post-RP BCR.Furthermore, snRNA RNU1-1/1-2 could serve as an independent prognostic factor for clinical RFS in post-RP BCR of HRPC cases where known prognostic factors (e.g., Gleason score) cannot distinguish between CR and non-CR patients.Our findings provide new insights into the involvement of snRNAs in prostate cancer progression.

Introduction
Prostate cancer (PC), the second most common male cancer, is an important global health issue; worldwide incidence and mortality rates have been increasing over the past couple of decades [1][2][3].Radical prostatectomy (RP), a definitive therapy for PC, is recommended for certain patients with high-risk PC (HRPC; prostate-specific antigen (PSA) ≥ 20 ng/mL, Gleason score (GS) ≥ 8, or clinical stage ≥ cT3a) [4,5].Although RP is highly effective, such patients have a higher risk of recurrence and progression after RP compared with patients exhibiting low-and intermediate-risk PC [6][7][8][9][10][11][12].After RP, a detectable serum PSA level of at least 0.2 ng/mL is considered indicative of biochemical recurrence (BCR); the presence of metastases on imaging after BCR is diagnostic of clinical recurrence (CR) [10,13,14].Among all patients with HRPC after RP, 46% experience BCR (designated as post-RP BCR in patients with HRPC); moreover, the 10-year PC-specific mortality rate among patients with HRPC who experience post-RP BCR can reach 9% [15].Both statistical and clinical indicators show that certain patients with HRPC who experience post-RP BCR have a high risk of CR [9,16,17].Thus, patients with HRPC who experience post-RP BCR require dedicated management and surveillance according to current risk stratification methods: pathological-grade group, PSA doubling time, and molecular imaging data [15,[18][19][20].An accurate prediction of such a patient subgroup that will develop metastatic progression (i.e., CR) or die of PC remains challenging.Additional prognostic biomarkers are required to reveal the subpopulation of patients with HRPC who may develop CR after post-RP BCR during extended follow-up and thus need second-line treatments (i.e., radiation/hormone therapy).
Formalin-fixed paraffin-embedded (FFPE) PC tissues obtained during RP constitute a critical resource in terms of the pathological diagnosis of PC.A pathological assessment of FFPE RP samples is important for guiding treatment decisions and predicting patient outcomes (e.g., PC-specific mortality) [21,22].A more accurate evaluation of GS using FFPE RP samples is important for PC risk management [23].FFPE RP samples serve as a valuable resource for molecular characterization of PC and biomarker discovery through in situ detection and/or extraction of FFPE biomolecules (e.g., nucleic acids, proteins, and metabolites), facilitating the comprehension of PC progression and aggressiveness [24][25][26][27].
Small nuclear RNAs (snRNAs) are small non-coding RNAs (~150 nucleotides in length) found in the nucleus.snRNAs serve as the RNA components of the spliceosome that recognizes 5 ′ and 3 ′ intron/exon junctions during intron splicing; they play essential roles in the processing of pre-mRNAs [28][29][30].snRNAs have recently received attention as potential biomarkers of certain types of cancer [31][32][33].However, very little is known about the diagnostic and prognostic utilities of snRNAs in PC.
The objective of this study was to identify new prognostic factors for CR in patients with HRPC who experience post-RP BCR, using FFPE RP samples.First, we performed a preliminary RNA sequencing analysis to comprehensively profile RNA expression in FFPE RP samples from patients with HRPC who developed CR after post-RP BCR.We found that many snRNAs were very abundant in preserved FFPE samples.Next, we used quantitative polymerase chain reaction (qPCR) to compare the expression levels of highly abundant snRNAs in FFPE RP samples between HRPC groups with and without CR after post-RP BCR.We evaluated the potential utilities of snRNAs as novel prognostic indicators of metastatic potential in patients with HRPC who experienced post-RP BCR.

Patient Selection and Study Design
Between October 2002 and January 2017, 633 patients were diagnosed with HRPC and underwent open/laparoscopic/robot-assisted RP at Nippon Medical School Hospital (NMSH) without any prior therapy.Follow-up was scheduled at least every 3 months after surgery.An increase of at least 0.2 ng/mL in the PSA value was considered to indicate BCR.All such patients received salvage radiotherapy or hormonal adjuvant therapy at the clinician's discretion.After RP, 178 patients experienced BCR (i.e., post-RP BCR in patients with HRPC).Of these, the numbers of patients who did and did not progress to CR were 24 and 154, respectively; CR was defined as metastatic disease confirmation on imaging studies (e.g., positron emission tomography/computed tomography and bone scintigraphy).Ineligible patients (for whom clinical or pathological information was inadequate, who underwent less than 5 years of follow-up after BCR, or whose tissue samples were inadequately stored) were excluded from the study.Finally, we enrolled 21 patients with CR (the CR group) and 46 non-CR patients with more than 5 years of followup after BCR (the non-CR group) when exploring candidate biomarkers for prediction of CR in patients with HRPC who experienced post-RP BCR.The patient selection criteria are presented in Figure 1, and the clinical characteristics of all patients are listed in Table 1.This study adhered to the 2013 Declaration of Helsinki and the principles of the Japanese Society of Pathology Ethics Committee.The NMSH Institutional Review Board approved this study (approval no.A-2020-049), and written informed consent was obtained from all patients.

Patient Selection and Study Design
Between October 2002 and January 2017, 633 patients were diagnosed with HRPC and underwent open/laparoscopic/robot-assisted RP at Nippon Medical School Hospital (NMSH) without any prior therapy.Follow-up was scheduled at least every 3 months after surgery.An increase of at least 0.2 ng/mL in the PSA value was considered to indicate BCR.All such patients received salvage radiotherapy or hormonal adjuvant therapy at the clinician's discretion.After RP, 178 patients experienced BCR (i.e., post-RP BCR in patients with HRPC).Of these, the numbers of patients who did and did not progress to CR were 24 and 154, respectively; CR was defined as metastatic disease confirmation on imaging studies (e.g., positron emission tomography/computed tomography and bone scintigraphy).Ineligible patients (for whom clinical or pathological information was inadequate, who underwent less than 5 years of follow-up after BCR, or whose tissue samples were inadequately stored) were excluded from the study.Finally, we enrolled 21 patients with CR (the CR group) and 46 non-CR patients with more than 5 years of follow-up after BCR (the non-CR group) when exploring candidate biomarkers for prediction of CR in patients with HRPC who experienced post-RP BCR.The patient selection criteria are presented in Figure 1, and the clinical characteristics of all patients are listed in Table 1.This study adhered to the 2013 Declaration of Helsinki and the principles of the Japanese Society of Pathology Ethics Committee.The NMSH Institutional Review Board approved this study (approval no.A-2020-049), and written informed consent was obtained from all patients.

Formalin-Fixed Paraffin-Embedded (FFPE) Radical Prostatectomy (RP) Specimens
A total of 67 FFPE RP specimens were obtained from the abovementioned 21 cases with CR and the 46 cases without CR in patients with HRPC who experienced post-RP BCR.RP samples were fixed in 20% formalin, sliced into approximately 3-5 mm thick slices perpendicular to the rectal surface from the apex of the prostate to the bladder neck side, and embedded in paraffin.Multiple hematoxylin-and-eosin-stained slides from these FFPE RP specimens were examined by pathologists of the Department of Clinical Pathology of NMSH to diagnose PC in accordance with the International Society of Urological Pathology (ISUP) grading system [13].All FFPE samples were stored at room temperature for between 5 and 20 years before RNA isolation was performed.For RNA extraction, sections with a thickness of 10 µm and, thus, volumes of approximately 10 mm 3 (e.g., four sections, each with an area of 250 mm 2 ) were collected from the PC regions of the FFPE blocks using a microtome (catalog no.TU213; Yamato Kohki Industrial Co., Ltd., Saitama, Japan).The clinicopathological data of the 67 prostate cancer cases included in this study are listed in Supplementary Table S1.

RNA-sequencing (RNA-seq)
First, we performed a preliminary RNA-seq analysis to comprehensively profile RNA expression, using three FFPE samples from the CR group that matched the relevant criteria (a DV200 value of at least 30% of total extracted RNA, a single peak at approximately 300 bp in the bioanalyzer electrophoretic diagram, and a total RNA library concentration of at least 4 nM).After rRNA depletion, cDNA libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (catalog no.E7760; New England Biolabs), in accordance with the manufacturer's protocol.All libraries were purified via the addition of AMPure XP magnetic beads (catalog no.A63811; Beckman Coulter, Pasadena, CA, USA); their qualities were assessed using an Agilent High Sensitivity DNA Kit (catalog no.5067-4626; Agilent Technologies) and a 2100 Bioanalyzer.Sequencing was performed on an Illumina HiSeq X Ten platform ( ) was used to count the numbers of reads that mapped to particular genes.

Statistical Analyses
All statistical analyses were performed with JMP software (version 13.2.0;SAS Institute, Cary, NC, USA).The characteristics of the two groups were compared using the Wilcoxon rank-sum test for continuous variables.Clinical recurrence-free survival (RFS) curves were generated by the Kaplan-Meier method and compared using the log-rank test.A multivariate Cox proportional hazards model was constructed with clinical RFS as the outcome variable; hazard ratios (for the CR group compared with the non-CR group), 95% confidence intervals, and p-values were calculated.qPCR data are expressed as means ± standard errors (SEs).A p-value < 0.05 was considered statistically significant.

RNA-seq of FFPE RP Samples from Patients with HRPC Who Developed CR after Post-RP BCR
First, we conducted an RNA-seq analysis of three FFPE RP samples from the CR group to determine which RNAs were stable and detectable in FFPE samples.It has been suggested that RNAs from clinical FFPE samples exhibit poor quality (e.g., they are degraded) [34,35].The clinicopathological characteristics of the three FFPE samples (nos. 1, 2, and 10) are listed in Supplementary Table S1.We obtained 6.98 million mapped reads, with a mean of 2.33 million mapped reads per sample.A total of 57,116 genes were detected via RNA-seq analysis (Supplementary Table S2 (gene analysis)); a summary is presented in Table 2. Protein-encoding RNA genes were the most abundant gene type detected via RNA-seq (i.e., 50.4% of all detected genes).In terms of non-coding RNA (ncRNA) genes, the relative abundances (in transcripts per million) of long ncRNA (lncRNA) genes and small ncRNA (i.e., miRNA, snRNA, and snoRNA) genes were 22.3% and 9.2% of all detected genes, respectively.The top 50 most highly expressed genes in FFPE samples are listed in Table 3.There were many snRNA and mitochondrial genes among the top 50 most highly expressed genes (Table 3).Genes of the nucleus and mitochondrion, as well as protein-encoding RNA genes in the cytosol, were well-preserved in FFPE samples [36,37].

Comparison of the Expression Levels of snRNAs between CR and Non-CR Groups Using qPCR
Intriguingly, many snRNA genes were included in the top 50 most highly expressed genes (Table 3), although snRNA genes constituted only 4.9% of all detected genes (in transcripts per million; Table 2).snRNAs primarily function to process pre-mRNAs in the nucleus [28][29][30], but their dysregulation has recently been reported in some cancers, indicating the potential importance of snRNAs as cancer biomarkers and therapeutic targets [31][32][33].Therefore, we investigated whether snRNAs in FFPE RP samples were prognostic factors for CR in patients with HRPC who experienced post-RP BCR.We focused on the three most highly expressed snRNA genes (i.e., RNU1-1/1-2, RNU4-1, and RNU4-2; Supplementary Table S3) and compared their expression levels between CR (n = 21) and non-CR (n = 46) groups via qPCR of the FFPE RP samples (Table 1 and Supplementary Table S1).As mentioned above, because the RNU1-1 and RNU1-2 genes share the same sequence, the two transcripts were regarded as a single gene (designated as RNU1-1/1-2).

Discussion
BCR is common, such that approximately 26% of all PC patients experience BCR within 15 years after RP (the primary definitive treatment) [15].BCR does not necessarily trigger CR; for patients with HRPC, BCR is associated with higher risks of distant metastasis and worse PC-specific mortality [15,17,41].Therefore, in patients with HRPC who experience post-RP BCR, complementary prognostic biomarkers are required to identify the approximately 10% of all patients who develop CR during extended follow-up [15].In this study, RNA-seq analysis demonstrated that many snRNA genes were very abundant in FFPE RP specimens from patients with HRPC who developed CR after post-RP BCR.The subsequent qPCR analysis of FFPE RP samples from patients with HRPC with and without CR after post-RP BCR revealed that the expression levels of snRNA RNU1-1/1-2 and RNU4-1 (designated as prostate cancer-associated snRNAs) were significantly higher in patients with CR than in patients without CR (Figure 2).PC-associated snRNA levels were significantly correlated with clinical RFS in patients with HRPC who experienced post-RP BCR; patients exhibiting high-level expression of the snRNAs experienced significantly shorter clinical RFS compared with patients exhibiting low-level expression (Figure 3).Correlations between snRNA levels and several clinicopathological factors (e.g., preoperative PSA level, ISUP Grade Group, and tumor stage) were also investigated (Table 4); the absence of correlations between the snRNAs and these factors, with the exception of preoperative PSA level, suggest that the PC-associated snRNAs could provide unique prognostic information.Furthermore, the multivariate survival analysis showed that snRNA RNU1-1/1-2 might serve as an independent prognostic factor for clinical RFS in patients with HRPC who experienced post-RP BCR (Table 5).The utility of RNU1-1/1-2 as a biomarker is reinforced by its independent nature, especially in cases where known prognostic factors cannot distinguish between CR and non-CR patients.
Until recently, only a few studies had analyzed cancer-associated snRNAs (e.g., U2 snRNA fragments [RNU2-1f]) [42][43][44][45].However, recent evidence indicates that aberrant snRNA expression induces tumorigenesis and cancer progression; snRNAs may serve as biomarkers of cancer prognosis and facilitate assessment of the treatment response [46][47][48][49].Recent studies of cancer-associated snRNAs have highlighted the significance of U1 snRNA (RNU1-1).Highly recurrent hotspot mutations (U1 r.3A>G mutations) of U1 snRNA were primarily associated with sonic hedgehog medulloblastoma [48].The U1 r.3A>G mutations drove 5 ′ cryptic alternative splicing, leading to inactivation of certain tumorsuppressor genes (e.g., PTCH1) [48].Moreover, a highly recurrent A>C somatic mutation (i.e., g.3A>C) in U1 has been observed in patients with chronic lymphocytic leukemia (CLL) and hepatocellular carcinoma (HCC) [47].This mutation created novel splice junctions and altered the splicing patterns of multiple genes, including known drivers of cancer (e.g., MSI2).The U1 g.3A>C mutation was associated with poor prognosis in patients exhibiting a more aggressive subtype of CLL.In addition to U1, N6-methyladenosine (m6A)-modified snRNAs (e.g., RNU6-2) were upregulated in HCC tissues compared with non-HCC tissues [31].Prognostic risk scores in patients with HCC, established using the Cancer Genome Atlas (TCGA) database based on the m6A-associated snRNA model, independently predicted overall survival in HCC patients.snRNAs (e.g., RNU6-1143P) were also associated with the overall survival of acute myeloid leukemia patients in the TCGA cohort [32].Low-level expression of RNU5E-1, a novel variant of U5 snRNA, was independently associated with improved tumor-free survival and long-term survival in patients with HCC [33].In the present study, we showed that aberrant expressions of snRNA RNU1-1/1-2 and RNU4-1 could serve as potential indicators of the prognosis in patients with HRPC who experience post-RP BCR.To our knowledge, this is the first report of PC-associated snRNAs.
The lncRNA PCA3 (specific to the prostate) is significantly overexpressed in PC [38,39], and its urine-based detection (i.e., the PCA3 test) is a valuable non-invasive method for PC diagnosis [40].However, the relationships of PCA3 status, aggressive features of PC, and treatment outcomes remain unclear; the evidence is conflicting [39].Merola et al. reported that higher urine PCA3 scores were associated with greater tumor aggressiveness (GS ≥ 7) [50].Conversely, Alshalalfa et al. reported that low-level PCA3 expression was associated with high Gleason grades (4 and 5) of biopsy and RP tissues; it was also correlated with a higher risk of metastasis and more aggressive PC after RP [51].We found no significant difference in PCA3 expression between FFPE RP tissues of the CR and non-CR groups (Figure 2D).Thus, the lncRNA PCA3 is unlikely to be involved in PC clinical recurrence after PR.In terms of the RNAs detected via RNA-seq of FFPE RP samples, several snRNA and snoRNA genes were among the top 50 most highly expressed genes (Table 3); however, the snRNA and snoRNA genes constituted only 4.9% and 2.0% of all genes (in transcripts per million), respectively (Table 2).snoRNAs, as well as snRNAs, are involved in tumorigenesis and cancer progression; snoRNAs have potential as useful diagnostic biomarkers and therapeutic targets in patients with various cancers [52][53][54].Further work is needed to determine whether aberrantly expressed snoRNAs are correlated with PC progression after RP.
Our work had some limitations.First, the sample size, especially for the CR group, was limited, and the research was conducted at a single institution.A multicenter cohort study is needed to validate our results.Second, we used FFPE samples.FFPE tissue processing and storage can trigger RNA degradation, fragmentation, and modification, all of which may affect the quality and reliability of RNA-seq and qPCR data [35,36,55].Thus, we used FFPE RNA extraction and library preparation methods that were specifically developed to improve the reliability of RNA-seq and qPCR data from FFPE-derived RNA samples [56,57].Although the results obtained from FFPE samples should be interpreted with caution, small ncRNAs (e.g., snRNAs and snoRNAs) are less likely to be adversely affected by FFPE sample preparation and storage compared with coding RNAs and lncRNAs [58].Additionally, the ways in which the PC-associated snRNAs identified in this study affect the molecular mechanisms of PC progression after RP require further investigation.

Conclusions
In conclusion, our RNA-seq and qPCR analyses, using FFPE RP specimens, yielded important information concerning novel, potentially prognostic factors for CR in patients with HRPC who experience post-RP BCR.In this study, we discovered that snRNA RNU1-1/1-2 was significantly upregulated in FFPE RP samples from patients with HRPC who developed CR after post-RP BCR.In such patients, there was a significant correlation between the snRNA RNU1-1/1-2 level and clinical RFS.snRNA RNU1-1/1-2 could serve as an independent prognostic factor for clinical RFS in patients with HRPC who experience post-RP BCR.Our findings offer new insights into the involvement of snRNAs in PC progression.

Table 1 .
Clinical characteristics of patients with high-risk prostate cancer (HRPC) with and without clinical recurrence (CR) after post-radical prostatectomy biochemical recurrence (post-RP BCR).

Table 1 .
Clinical characteristics of patients with high-risk prostate cancer (HRPC) with and without clinical recurrence (CR) after post-radical prostatectomy biochemical recurrence (post-RP BCR).

Table 3 .
The top 50 most highly expressed genes detected by RNA-seq of FFPE RP samples from patients with HRPC who developed CR after post-RP BCR (gene analysis).

Table 5 .
Multivariate Cox regression analysis of prognostic factors contributing to clinical RFS in patients with HRPC who experienced post-RP BCR.

Table 5 .
Multivariate Cox regression analysis of prognostic factors contributing to clinical RFS in patients with HRPC who experienced post-RP BCR.